NLS-RARα蛋白相互作用蛋白的筛选与验证(英文)

Screening and Identification of The Proteins Interacting With NLS-RARα Protein

急性早幼粒细胞白血病(APL)具有特征性染色体易位,产生的PML-RARα融合基因在其发生发展中有重要作用.PML-RARα融合蛋白在细胞内被中性粒细胞弹性蛋白酶(neutrophil elastase,NE)切割为PML突变蛋白(核定位信号NLS缺失)和RARα突变体(NLS-RARα,包含有PML的核定位信号),这两段蛋白质在APL的发生中可能具有重要作用.为进一步研究NLS-RARα的生物学功能,运用酵母双杂交技术在白血病cDNA文库中筛选与其作用的蛋白质.首先PCR技术扩增NLS-RARα编码序列,克隆至诱饵载体pGBKT7,测序鉴定后将其转化酵母AH109.免疫印迹检测到诱饵蛋白表达后,将含有诱饵载体的AH109与含有白血病cDNA文库的酵母Y187交配,在含有X-α-gal的营养缺陷性培养基上选择和筛选二倍体酵母.经回转实验和测序分析验证得到8个与NLS-RARα相互作用的蛋白质.为进一步验证这些相互作用,克隆其中的JTV-1蛋白,利用间接免疫荧光,GSTpull-down和免疫共沉淀技术成功验证了它与NLS-RARα的相互作用.为进一步探讨APL的发生机制提供了新的线索.

Acute promyelocytic leukemia （APL） is characterized by the generation of the prototypic promyelocytic leukemia-retinoic acid receptor alpha （PML-RARα）, an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line, PML-RARα is cleaved by neutrophil elastase （NE） to produce the mutational PML [nuclear localization signal （NLS） deletion] and RARα （NLS-RARα, containing NLS of PML）, both of which may play an important role in APL pathogenesis. The yeast two-hybrid technique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7 vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmid was proved by Western-blot. The transformed yeast AH109 was mated with yeast Y187 （containing leukemia cDNA library plasmids pACT2） in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After being reintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among which one containing JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence, GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration of NLS-RARα signaling to APL.